CN110904250A - Multiple fluorescent quantitative PCR primer, kit and detection method for detecting multiple bacteria - Google Patents
Multiple fluorescent quantitative PCR primer, kit and detection method for detecting multiple bacteria Download PDFInfo
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Abstract
The embodiment of the invention discloses a multiple fluorescence quantitative PCR primer, a kit and a detection method for detecting multiple bacteria, which comprise a primer pair and a probe designed aiming at 16SrDNA of bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri and akkermansia bacteria, wherein the nucleotide sequences of the primer pair of the bifidobacterium longum, the bifidobacterium animalis and the bifidobacterium breve are shown as SEQ ID NO. 1 and SEQ ID NO. 2. The invention also discloses a multiple fluorescence quantitative PCR detection kit containing the PCR primer and the probe. The multiple fluorescent quantitative PCR primers and probes for detecting multiple bacteria provided by the embodiment of the invention are synthesized according to specific sequences which are subjected to multiple experiments and designed, and the specific primers and probes which are optimized through the experiments can ensure better specificity during amplification and reduce mutual interference of the primer probes in the same reaction system.
Description
Technical Field
The embodiment of the invention relates to the technical field of microbial detection, in particular to a multiple fluorescent quantitative PCR primer, a kit and a detection method for detecting multiple bacteria.
Background
Human intestinal microorganisms have a close relationship with human health, and the proportion of some important types of bacteria has an important influence on the human health. The content and the proportion of some important strains in the intestinal microorganisms are detected, so that the method has certain significance for judging certain health conditions of the examined person and assisting treatment. Currently, common detection methods for the number and the type of bacteria include: 1. traditional selective medium culture: the method mainly comprises the steps of culturing specific bacteria in a sample through a specific culture medium, and reflecting the number of the bacteria in the sample through the number of colonies. The method is a classical and common method, and the detection result is more accurate. The disadvantages of this method are: the culture period is long, and generally needs several days; the detection flux is low, a lot of bacteria with strict requirements on culture conditions are difficult to culture, the identification precision is general, and subspecies or mutant strains cannot be accurately distinguished under most conditions. Unknown strains cannot be found, and multiple strains cannot be detected in the same culture medium in most cases. 2. Biochemical identification: the specific chemical substance generated by the strain to be detected is utilized to detect the existence of the corresponding strain in the sample through specific chemical reaction. The detection time is shorter than that of a culture medium culture method, and the flux is improved. However, there are also some problems that the accuracy of detection is limited and it is impossible to distinguish between subspecies and mutants. In addition, many bacteria cannot produce specific chemical substances and cannot be detected by the method; in many cases, it is impossible to detect a plurality of bacteria by the same reaction. 3. Fluorescent quantitative PCR: designing probes and primers according to the specific DNA sequence of the corresponding strain, and detecting the DNA of the strain to confirm the existence or content of the strain. The detection method has short detection time and high detection sensitivity, and only a few hours are needed for DNA extraction and amplification; the detection precision is high, and the detection can be accurate to subspecies or mutant strains; and various bacteria can be detected by one reaction. The disadvantage is that the specific DNA sequence of the bacteria to be detected needs to be known, the bacteria with unknown sequence or mutant strains cannot be detected, and the detection and quantification accuracy can be influenced if SNP mutation occurs on the DNA of the bacteria at the primer or probe. 4. NGS second generation sequencing: and (3) establishing a library for DNA extracted from the sample by using a metagenome or 16S mode, and obtaining the species and proportion of various bacteria through next-generation sequencing and bioinformatics analysis. The method has the advantages of high detection flux, high detection sensitivity, accurate detection result and capability of detecting some unknown bacteria by means of metagenome. The defects are that the detection period is relatively long and the detection cost is high.
There are many fields in which the detection of a few specific known bacteria in a sample is required. It is necessary to detect whether or not these bacteria are contained in the sample and the respective contents of these bacteria in the sample in a short time. Most of the tests have higher requirements on the detection period and the detection precision, the time for obtaining the detection result from the detection of the sample generally needs to be within one day or even shorter, and the bacteria also need to be precise to the seed or subspecies level. Therefore, the fluorescence quantitative PCR is a common detection means, which can obtain the detection result within several hours, and can accurately obtain the number of the bacteria in the sample at the seed and subspecies level.
The current relatively broad fluorescence quantitative method for detection is generally to detect a bacterium in a tube reaction system. The detection flux is relatively low, and the detection cost is relatively high. If a multiplex PCR amplification and detection technology is used, multiple bacteria can be detected in one tube reaction, so that the detection flux can be greatly improved, and the detection cost is reduced.
However, the difficulty of multiplex PCR is much higher than that of ordinary PCR, and the following defects exist: 1. specificity of primers and probes: because some bacteria in the same genus have higher DNA sequence homology, if the specificity of a primer and a probe is not high, non-specific amplification can be caused, and the sequences of the bacteria with similar DNA sequences are amplified, so that false positive is caused, the detection accuracy is influenced, and the number of the detected target bacteria is increased. 2. Mutual interference of primer probes: for the fluorescent quantitative PCR to detect a plurality of bacteria in one reaction system, the proper primers and probes are especially important because the primers and probes exist in the same reaction system and are easy to interfere with each other to cause inaccuracy of detection results. 3. Amplification efficiency of the primers: if the amplification efficiency of the primers is different, when amplification is performed simultaneously in the same reaction system, imbalance of initial DNA amplification of a sample can be caused due to different amplification efficiencies among the primers, and some bacteria with low content or bacteria with low primer amplification efficiency cannot compete with the primers with high content or high amplification efficiency, so that the amplification products of the bacteria are fewer, fluorescent signals are weaker, false negative results are caused, and the accuracy of quantification is influenced.
Disclosure of Invention
Therefore, the embodiment of the invention provides a multiple fluorescent quantitative PCR primer, a kit and a detection method for detecting multiple bacteria, so as to solve the problem that the detection efficiency of the multiple bacteria is low due to poor detection specificity in the prior art.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:
a multiplex fluorescence quantitative PCR primer and a probe for detecting various bacteria comprise a primer pair and a probe designed aiming at 16SrDNA of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri and Ackermansonii, wherein the nucleotide sequences of the primer pair of Bifidobacterium longum, Bifidobacterium animalis and Bifidobacterium breve are shown as SEQ ID NO. 1 and SEQ ID NO. 2;
the sequence of the bifidobacterium longum nucleotide probe is shown as SEQ ID NO. 3, the sequence of the bifidobacterium animalis nucleotide probe is shown as SEQ ID NO. 4, and the sequence of the bifidobacterium breve nucleotide probe is shown as SEQ ID NO. 5;
the nucleotide sequences of the primer pair of the lactobacillus rhamnosus are shown as SEQ ID NO. 6 and SEQ ID NO. 7, and the nucleotide probe sequence of the lactobacillus rhamnosus is shown as SEQ ID NO. 8;
the nucleotide sequences of the primer pair of the lactobacillus reuteri are shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the nucleotide probe sequence of the lactobacillus reuteri is shown as SEQ ID NO. 11;
the nucleotide sequences of the primer pair of the akkermansia are shown as SEQ ID NO. 12 and SEQ ID NO. 13, and the nucleotide probe sequence of the akkermansia is shown as SEQ ID NO. 14;
the nucleotide sequences of the primer pair of the lactobacillus acidophilus are shown as SEQ ID NO. 15 and SEQ ID NO. 16, and the nucleotide probe sequence of the lactobacillus acidophilus is shown as SEQ ID NO. 17;
the nucleotide sequence of the lactobacillus plantarum primer pair is shown as SEQ ID NO. 18 and SEQ ID NO. 19, and the lactobacillus plantarum nucleotide probe sequence is shown as SEQ ID NO. 20.
The embodiment of the invention also provides a multiplex fluorescence quantitative PCR detection kit for detecting multiple bacteria, and the PCR detection reagent comprises the multiplex fluorescence quantitative PCR primer and the probe for detecting the multiple bacteria.
The multiplex fluorescence quantitative PCR detection kit for detecting various bacteria of the embodiment of the invention also comprises a kit containing Mg2+The PCR reaction buffer solution, dNTP, and PCR amplification enzyme of (1).
Preferably, the detection kit is used for simultaneously detecting bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri and akkermansia.
Preferably, the PCR primers and probes are divided into a first component, a second component, and a third component;
wherein the first component comprises primers and probes for the bifidobacterium longum, bifidobacterium breve and bifidobacterium animalis;
the second component comprises primers and probes of the lactobacillus rhamnosus and the lactobacillus reuteri;
the third component comprises primers and probes of Ackermanella, Lactobacillus acidophilus and Lactobacillus plantarum.
In another aspect, the present invention further provides a multiplex fluorescence quantitative PCR method for detecting multiple bacteria, which includes a process of performing multiplex fluorescence quantitative PCR using 16SrDNA of bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri, and akkermansia as a template, and the PCR primers and probes described above.
Preferably, the PCR primers and probes are divided into a first component, a second component, and a third component;
wherein the first component comprises primers and probes for the bifidobacterium longum, bifidobacterium breve and bifidobacterium animalis;
the second component comprises primers and probes of the lactobacillus rhamnosus and the lactobacillus reuteri;
the third component comprises primers and probes of Ackermanella, Lactobacillus acidophilus and Lactobacillus plantarum.
Preferably, the multiple fluorescent quantitative PCR reaction process comprises decontamination at 50 ℃ for 3min, 1cycle, pre-denaturation at 95 ℃ for 15min, denaturation at 95 ℃ for 20s and 35 cycles, annealing at 60 ℃ for 40s, and extension at 60 ℃ for 40 s.
The embodiment of the invention has the following advantages:
the multiple fluorescent quantitative PCR primers and probes for detecting multiple bacteria provided by the embodiment of the invention are synthesized according to specific sequences which are subjected to multiple experiments and designed, and the specific primers and probes which are optimized through the experiments can ensure better specificity during amplification and reduce the mutual interference of the primer probes in the same reaction system; the kit prepared by the primer and the probe can detect 8 thalli simultaneously, and has the advantages of high flux, accurate detection result and good specificity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
Fig. 1 is a fluorescence quantitative graph of a first component for detecting bifidobacterium, provided in an embodiment of the present invention, wherein curve a represents bifidobacterium animalis, curve B represents bifidobacterium longum, and curve C represents bifidobacterium breve;
FIG. 2 is a fluorescence quantitative graph of a second component for detecting Lactobacillus rhamnosus and Lactobacillus reuteri according to an embodiment of the present invention, wherein curve A is Lactobacillus rhamnosus and curve B is Lactobacillus reuteri;
fig. 3 is a fluorescence quantitative graph for detecting ackerman, lactobacillus acidophilus and lactobacillus plantarum by the third component according to the embodiment of the present invention, wherein curve a is lactobacillus plantarum, curve B is lactobacillus acidophilus, and curve C is ackerman.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment of the invention provides a multiplex fluorescence quantitative PCR primer and a probe for detecting multiple bacteria, and particularly the PCR primer and the probe are used for detecting 8 bacteria such as bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri, akkermansia and the like in human intestinal tracts, and the primer and the probe are designed according to respective specific 16SrDNA sequences of the 8 bacteria and are tested. By continuously designing and optimizing PCR primers and probes according to the detection result, the PCR reaction system is adjusted in a targeted manner. A group of fluorescence quantitative PCR primers and probe combinations which are ideal in the aspects of specificity, mutual interference and amplification efficiency of the primers and probes are designed, as shown in Table 1, the multiple fluorescence quantitative PCR primers and probes for detecting various bacteria in the embodiment of the invention comprise primer pairs and probes designed for 16SrDNA of bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri and akkermansia, wherein the nucleotide sequences of the primer pairs of the bifidobacterium longum, the bifidobacterium animalis and the bifidobacterium breve are shown as SEQ ID NO 1 and SEQ ID NO 2; the sequence of the bifidobacterium longum nucleotide probe is shown as SEQ ID NO. 3, the sequence of the bifidobacterium animalis nucleotide probe is shown as SEQ ID NO. 4, and the sequence of the bifidobacterium breve nucleotide probe is shown as SEQ ID NO. 5; the nucleotide sequence of the primer pair of the lactobacillus rhamnosus is shown as SEQ ID NO. 6 and SEQ ID NO. 7, and the nucleotide probe sequence of the lactobacillus rhamnosus is shown as SEQ ID NO. 8; the nucleotide sequence of the primer pair of the lactobacillus reuteri is shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the nucleotide probe sequence of the lactobacillus reuteri is shown as SEQ ID NO. 11; the nucleotide sequences of the primer pair of the akkermansia are shown as SEQ ID NO. 12 and SEQ ID NO. 13, and the nucleotide probe sequence of the akkermansia is shown as SEQ ID NO. 14; the nucleotide sequence of the primer pair of the lactobacillus acidophilus is shown as SEQ ID NO. 15 and SEQ ID NO. 16, and the nucleotide probe sequence of the lactobacillus acidophilus is shown as SEQ ID NO. 17; the nucleotide sequence of the lactobacillus plantarum primer pair is shown as SEQ ID NO. 18 and SEQ ID NO. 19, and the lactobacillus plantarum nucleotide probe sequence is shown as SEQ ID NO. 20.
TABLE 1
The primer and probe design reference sequences are both from the NCBI website Genebank database, and the primer and probe are synthesized by Beijing Ongchoku Biotechnology Co.
Example 2
Dividing the PCR primers and probes prepared according to the design of example 1 into a first component, a second component and a third component, as shown in tables 2-4; wherein the first component comprises primers and probes of the bifidobacterium longum, the bifidobacterium breve and the bifidobacterium animalis as well as a buffer solution and an enzyme required by a PCR reaction system; the second component comprises primers of the lactobacillus rhamnosus and the lactobacillus reuteri, and a buffer solution and an enzyme required by a probe PCR reaction system; the third component comprises primers and probes of Ackermanella, Lactobacillus acidophilus and Lactobacillus plantarum and a buffer solution and an enzyme required by a PCR reaction system.
TABLE 2
TABLE 3
TABLE 4
The reaction procedure of the multiplex quantitative PCR method according to the present example is shown in table 5.
TABLE 5
According to the embodiment of the invention, through multiple designs and experiments, each component corresponds to one reaction tube, and 8 bacteria of bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri and akkermansia are respectively detected in a PCR reaction system of 3 reaction tubes, so that the detection flux can be obviously improved, and the detection cost can be obviously reduced.
Test examples
Respectively extracting 8 standard strains of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus roseusDNA of Lactobacillus helveticus and Ackermanella, and detecting the concentration of the DNA; a reaction solution is prepared according to a reaction system of fluorescent quantitative PCR, and a prescribed amount of DNA template is added to the PCR reaction system based on the measured DNA concentration. In the PCR reaction system, the dosage of the DNA template is 25-50ng, the dosages of the primer and the probe are both 2nM, the concentration of dNTPs is 2mM/mL, and Mg2+The concentration was 20 mM/mL.
The products were amplified according to the amplification procedure set forth in Table 5, and the respective fluorescent signals of each round of amplification were detected. And respectively carrying out multiplex quantitative fluorescent PCR detection on the 8 standard strains by using the first component, the second component and the third component so as to detect the specificity of the first component, the second component and the third component. The information on the standard strains used is shown in Table 6.
TABLE 6
The reaction was carried out according to the reaction procedure shown in Table 5, and the results of multiplex quantitative fluorescent PCR specificity test for 8 standard strains are shown in Table 7.
TABLE 7
As shown in fig. 1 to fig. 3, the results of detecting bifidobacterium with the first component, lactobacillus rhamnosus and lactobacillus reuteri with the second component, and lactobacillus acidophilus and lactobacillus plantarum with the third component are analyzed to obtain the specific contents of various bacteria.
Example of clinical test
The multiplex fluorescent quantitative PCR detection kit for detecting various bacteria of the embodiment of the invention is used for testing 50 samples of children and adults, and the system and the reaction conditions for extracting template DNA and multiplex fluorescent quantitative PCR are shown in the test embodiment. The results of the tests on the samples of children and adults are shown in Table 8. The test samples are given by cooperative medical units, all the samples are stored in a refrigerator at the temperature of-80 ℃, the bacteria DNA is extracted by using a Siranolong biological NP968-S nucleic acid extractor and a matched reagent thereof, the concentration of the extracted nucleic acid is determined, and the detection is carried out by using a multiple fluorescence quantitative PCR system. 16srDNA sequencing is carried out on the same sample by using an Illumina miseq sequencer synchronously, and the content of various bacteria in the sample on the genus level is detected. Meanwhile, the method reported in the reference literature, and the fluorescent dye, can only detect one kind of bacteria in each tube.
TABLE 8
The results of the detection of the genus level content of each sample by using a Miseq sequencer of Illumina are shown in Table 9, and the method can only detect the genus level content ratio and cannot distinguish the accurate content of each species in the genus.
TABLE 9
The content of each bacterium in the sample is detected by using fluorescent quantitative PCR, and the result is as follows: due to the different detection methods, the result has a certain difference with the multiple fluorescence quantitative detection result of the embodiment of the invention.
As can be seen from the detection results in tables 8-9, the multiplex fluorescence quantitative PCR detection of the embodiment of the invention has accurate result, high flux and good specificity.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (8)
1. A multiplex fluorescence quantitative PCR primer and a probe for detecting various bacteria are characterized by comprising a primer pair and a probe designed aiming at 16SrDNA of bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri and akkermansia, wherein the nucleotide sequences of the primer pair of the bifidobacterium longum, the bifidobacterium animalis and the bifidobacterium breve are shown as SEQ ID NO. 1 and SEQ ID NO. 2;
the sequence of the bifidobacterium longum nucleotide probe is shown as SEQ ID NO. 3, the sequence of the bifidobacterium animalis nucleotide probe is shown as SEQ ID NO. 4, and the sequence of the bifidobacterium breve nucleotide probe is shown as SEQ ID NO. 5;
the nucleotide sequences of the primer pair of the lactobacillus rhamnosus are shown as SEQ ID NO. 6 and SEQ ID NO. 7, and the nucleotide probe sequence of the lactobacillus rhamnosus is shown as SEQ ID NO. 8;
the nucleotide sequences of the primer pair of the lactobacillus reuteri are shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the nucleotide probe sequence of the lactobacillus reuteri is shown as SEQ ID NO. 11;
the nucleotide sequences of the primer pair of the akkermansia are shown as SEQ ID NO. 12 and SEQ ID NO. 13, and the nucleotide probe sequence of the akkermansia is shown as SEQ ID NO. 14;
the nucleotide sequences of the primer pair of the lactobacillus acidophilus are shown as SEQ ID NO. 15 and SEQ ID NO. 16, and the nucleotide probe sequence of the lactobacillus acidophilus is shown as SEQ ID NO. 17;
the nucleotide sequence of the lactobacillus plantarum primer pair is shown as SEQ ID NO. 18 and SEQ ID NO. 19, and the lactobacillus plantarum nucleotide probe sequence is shown as SEQ ID NO. 20.
2. A multiplex fluorescence quantitative PCR detection kit for detecting various bacteria is characterized in that,
the PCR detection reagent and the multiplex fluorescent quantitative PCR primer and probe for detecting various bacteria, which are disclosed by the claim 1.
3. The multiplex quantitative fluorescent PCR detection kit for detecting multiple bacteria according to claim 2, further comprising Mg2+The PCR reaction buffer solution, dNTP, and PCR amplification enzyme of (1).
4. The multiplex fluorescent quantitative PCR detection kit for detecting a plurality of bacteria according to claim 2,
the detection kit is used for simultaneously detecting bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri and akkermansia.
5. The multiplex fluorescent quantitative PCR detection kit for detecting a plurality of bacteria according to claim 2,
the PCR primers and the probes are divided into a first component, a second component and a third component;
wherein the first component comprises primers and probes for the bifidobacterium longum, bifidobacterium breve and bifidobacterium animalis;
the second component comprises primers and probes of the lactobacillus rhamnosus and the lactobacillus reuteri;
the third component comprises primers and probes of Ackermanella, Lactobacillus acidophilus and Lactobacillus plantarum.
6. A multiplex fluorescent quantitative PCR method for detecting multiple bacteria, which is characterized in that the method comprises the process of carrying out multiplex fluorescent quantitative PCR by using 16SrDNA of bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus reuteri and akkermansia as templates and the PCR primer and probe of claim 1.
7. The method for multiplex fluorescent quantitative PCR for detecting a plurality of bacteria according to claim 6,
the PCR primers and the probes are divided into a first component, a second component and a third component;
wherein the first component comprises primers and probes for the bifidobacterium longum, bifidobacterium breve and bifidobacterium animalis;
the second component comprises primers and probes of the lactobacillus rhamnosus and the lactobacillus reuteri;
the third component comprises primers and probes of Ackermanella, Lactobacillus acidophilus and Lactobacillus plantarum.
8. The method for multiplex fluorescent quantitative PCR for detecting a plurality of bacteria according to claim 6,
the multiple fluorescent quantitative PCR reaction process comprises the steps of decontamination at 50 ℃ for 3min, 1cycle, pre-denaturation at 95 ℃ for 15min, denaturation at 95 ℃ for 20s and 35 cycles, annealing at 60 ℃ for 40s, and extension at 60 ℃ for 40 s.
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